NOX2 Deficiency Research Articles

Female X-Chromosome Mosaicism for NOX2 Deficiency Presents Unique Inflammatory Phenotype and Improves Outcome in Polymicrobial Sepsis

Cellular X-chromosome mosaicism, which is unique to females, may be advantageous during pathophysiological challenges compared with the single X-chromosome machinery of males, and it may contribute to gender dimorphism in the inflammatory response. We tested the hypothesis of whether cellular mosaicism for the X-linked gp91phox (NOX2) deficiency, the catalytic component of the superoxide anion-generating NADPH oxidase complex, is advantageous during polymicrobial sepsis. Deficient, wild-type (WT), and heterozygous/mosaic mice were compared following polymicrobial sepsis initiated by cecal ligation and puncture. Compared with WT littermates, sepsis-induced mortality was improved in deficient mice, as well as in mosaic animals carrying both deficient and WT phagocyte subpopulations. In contrast, blood bacterial counts were greatest in deficient mice. Consistent with poor survival, WT mice also showed the most severe organ damage following sepsis. In mosaic animals, the deficient neutrophil subpopulations displayed increased organ recruitment and elevated CD11b membrane expression compared with WT neutrophil subpopulations within the same animal. The dynamics of sepsis-induced blood and organ cytokine content and WBC composition changes, including lymphocyte subsets in blood and bone marrow, showed differences among WT, deficient, and mosaic subjects, indicating that mosaic mice are not simply the average of the deficient and WT responses. Upon oxidative burst, interchange of oxidants between WT and deficient neutrophil subpopulations occurred in mosaic mice. This study suggests that mice mosaic for gp91phox expression have multiple advantages compared with WT and deficient mice during the septic course.

Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b+ and Th/Treg cells

Emerging evidence indicates that NADPH oxidase (NOX) and its reactive oxygen species (ROS) products modulate a variety of cellular events, including proliferation, differentiation, and apoptosis. In this study, we investigated the functions of NOX2 and ROS in immune modulation using NOX2 knockout (KO) mice. Interestingly, NOX2 KO mice spontaneously developed arthritis with onset at 6-7 wk of age and high incidence (60%) at 15-18 wk of age. Arthritis severity in NOX2 KO mice was proportionally increased with age and higher in females than in males. Bone destruction was confirmed by microcomputed tomography scanning and histological analyses of joints. Inflammatory factors, including TNF-α, IL-1β, and RANKL, and serum level of anti-type II collagen IgG were significantly increased in NOX2 KO mice. In addition, NOX2 deficiency perturbed the immune system upon aging. NOX2 KO mice demonstrated preferred development of CD11b+Gr-1+ myeloid cells with profound production of proinflammatory cytokines and augmented expression of IL-17 through the activation of STAT3 and RORγt in vivo. NOX2 deficiency increased differentiation of effector Th cells in vitro and decreased CD25+FoxP3+ Treg cells both in vitro and in vivo. Furthermore, adoptive transfer of NOX2-deficient CD4(+) T cells into RAG KO mice increased arthritic inflammation compared with WT cells. These results demonstrated that NOX2 deficiency affected the development of CD11b+ myeloid cells and Th17/Treg cells, and thus promoted inflammatory cytokine production and inflammatory arthritis development, strongly supporting a crucial role for ROS generation in the modulation of Th17/Treg cell development and its related inflammatory immune response upon aging.

Nox2-derived reactive oxygen species contribute to hypercholesterolemia-induced inhibition of neovascularization: Effects on endothelial progenitor cells and mature endothelial cells

Background Hypercholesterolemia has been associated with impaired angiogenesis and reduced blood flow recuperation after ischemia. However, the precise mechanisms involved are unknown. Here we investigated the role of Nox2-derived reactive oxygen species (ROS) in the modulation of neovascularization by hypercholesterolemia. Methods and results Mice deficient for the Nox2-containing NADPH oxidase (Nox2 −/−) and control mice (Nox2 +/+) were put on a high cholesterol diet (HCD) for a total of 15 weeks. After three months, hindlimb ischemia was surgically induced by femoral artery removal. Nox2 expression and oxidative stress levels in ischemic tissues were significantly increased by HCD in control mice, but not in Nox2 −/− mice. Nox2 −/− mice were also protected against hypercholesterolemia-induced impairment of neovascularization, as demonstrated by faster blood flow recovery after ischemia and increased capillary density in ischemic muscles. Nox2 deficiency was associated with preserved activity of eNOS in ischemic tissues, and improved activity of endothelial progenitor cells (EPCs). In vitro, HUVECs treated with the NADPH oxidase inhibitor apocynin or endothelial cells isolated from the aorta of Nox2 −/− mice exhibited reduced ROS formation following exposure to oxLDL. This was associated with improved nitric oxide (NO) bioavailability and protection against oxLDL-induced inhibition of angiogenic activities. Conclusions Nox2-containing NADPH oxidase deficiency protects against hypercholesterolemia-induced impairment of neovascularization. The potential mechanisms involved include reduced ROS formation, preserved activation of angiogenic signals, and improved functional activities of EPCs and mature endothelial cells.

Chronic Fine Particulate Matter Exposure Induces Systemic Vascular Dysfunction via NADPH Oxidase and TLR4 Pathways

Chronic exposure to ambient air-borne particulate matter of < 2.5 μm (PM₂.₅) increases cardiovascular risk. The mechanisms by which inhaled ambient particles are sensed and how these effects are systemically transduced remain elusive. To investigate the molecular mechanisms by which PM₂.₅ mediates inflammatory responses in a mouse model of chronic exposure. Here, we show that chronic exposure to ambient PM₂.₅ promotes Ly6C(high) inflammatory monocyte egress from bone-marrow and mediates their entry into tissue niches where they generate reactive oxygen species via NADPH oxidase. Toll-like receptor (TLR)4 and Nox2 (gp91(phox)) deficiency prevented monocyte NADPH oxidase activation in response to PM₂.₅ and was associated with restoration of systemic vascular dysfunction. TLR4 activation appeared to be a prerequisite for NAPDH oxidase activation as evidenced by reduced p47(phox) phosphorylation in TLR4 deficient animals. PM₂.₅ exposure markedly increased oxidized phospholipid derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in bronchioalveolar lavage fluid. Correspondingly, exposure of bone marrow-derived macrophages to oxPAPC but not PAPC recapitulated effects of chronic PM₂.₅ exposure, whereas TLR4 deficiency attenuated this response. Taken together, our findings suggest that PM₂.₅ triggers an increase in oxidized phospholipids in lungs that then mediates a systemic cellular inflammatory response through TLR4/NADPH oxidase-dependent mechanisms.

Enhancement of Allergen-induced Airway Inflammation by NOX2 Deficiency

BackgroundNADPH oxidase (NOX) modulates cell proliferation, differentiation and immune response through generation of reactive oxygen species. Particularly, NOX2 is recently reported to be important for regulating Treg cell differentiation of CD4+ T cells.MethodsWe employed ovalbumin-induced airway inflammation in wild-type and NOX2-deficient mice and analyzed tissue histopathology and cytokine profiles.ResultsWe investigated whether NOX2-deficiency affects T cell-mediated airway inflammation. Ovalbumin injection which activates T cell-mediated allergic response increased airway inflammation in wild-type mice, as evidenced by increased immune cell infiltration, allergic cytokine expression, and goblet cell hyperplasia in the lung. Interestingly, NOX2 knockout (KO) mice were more susceptible to allergen-induced lung inflammation compared to wild-type mice. Immune cells including neutrophils, lymphocytes, macrophages, and eosinophils were drastically infiltrated into the lung of NOX2 KO mice and mucus secretion was substantially increased in deficiency of NOX2. Furthermore, inflammatory allergic cytokines and eotaxin were significantly elevated in NOX2 KO mice, in accordance with enhanced generation of inflammatory cytokines interleukin-17 and interferon-γ by CD4+ T cells.ConclusionThese results indicate that NOX2 deficiency favorably produces inflammatory cytokines by T cells and thus increases the susceptibility to severe airway inflammation.

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

Endoplasmic reticulum (ER)-induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress-apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA-dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.

The NADPH Oxidase NOX2 Controls Glutamate Release: A Novel Mechanism Involved in Psychosis-Like Ketamine Responses

Subanesthetic doses of NMDA receptor antagonist ketamine induce schizophrenia-like symptoms in humans and behavioral changes in rodents. Subchronic administration of ketamine leads to loss of parvalbumin-positive interneurons through reactive oxygen species (ROS), generated by the NADPH oxidase NOX2. However, ketamine induces very rapid alterations, in both mice and humans. Thus, we have investigated the role of NOX2 in acute responses to subanesthetic doses of ketamine. In wild-type mice, ketamine caused rapid (30 min) behavioral alterations, release of neurotransmitters, and brain oxidative stress, whereas NOX2-deficient mice did not display such alterations. Decreased expression of the subunit 2A of the NMDA receptor after repetitive ketamine exposure was also precluded by NOX2 deficiency. However, neurotransmitter release and behavioral changes in response to amphetamine were not altered in NOX2-deficient mice. Our results suggest that NOX2 is a major source of ROS production in the prefrontal cortex controlling glutamate release and associated behavioral alterations after acute ketamine exposure. Prolonged NOX2-dependent glutamate release may lead to neuroadaptative downregulation of NMDA receptor subunits.

NOX2 Deficiency Protects Against Streptozotocin-Induced β-Cell Destruction and Development of Diabetes in Mice

OBJECTIVEThe role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice.RESEARCH DESIGN AND METHODSFive groups of mice—wild-type (WT), NOX2−/−, WT treated with apocynin, and WT adoptively transferred with NOX2−/− or WT splenocytes—were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2−/− pancreatic islets were treated with cytokines for 48 h.RESULTSSignificantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2−/− mice and in WT mice adoptively transferred with NOX2−/− splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2−/− mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2−/− compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2−/− compared with WT splenocytes.CONCLUSIONSNOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis.

NADPH oxidases and atherosclerosis: unraveling the details

NADPH oxidases and atherosclerosis: unraveling the details

Abstract 4909: Nox2-Containing NADPH Oxidase Deficiency Protects Against Age-Dependent Inhibition of Neovascularization

Background: We have previously shown that aging is associated with an impaired angiogenic response following ischemia. Because aging leads to an increased generation of reactive oxygen species (ROS), here we investigated the role of Nox2-containing NADPH oxidase ROS generation for the modulation of neovascularization by aging. Methods: Hindlimb ischemia was surgically induced by femoral artery removal in young (5–7 weeks) and older (9 months) Nox2 −/− and control (Nox2 +/+ ) mice. Results: We found that blood flow recovery is significantly reduced in older compared to younger mice at day 21 after surgery (Doppler flow ratios: 0.51± 0.05 vs. 0.72± 0.05; p&lt;0.05). At the microvascular level, capillary density was also significantly reduced in ischemic muscles of older animals (53.4± 6.3 capillaries/field vs. 82.5± 4.9 capillaries/field; p&lt;0.001). This was associated with a significant increase of oxidative stress levels (nitrotyrosine immunostaining) in ischemic tissues of older animals. Importantly, we found that Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation in older animals. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. Here we found that the functional activities of EPCs (adhesion to HUVECs, migration) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPCs functional activities in older animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels (DCF-DA, DHE) in EPCs that is not present in Nox2-deficient animals. Conclusion: Nox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.

Abstract 5325: Inherited Human Nox2 Deficiency is Associated With Impaired Isoprostane Formation and Reduced Platelet Recruitment

Platelet isoprostane PGF2 α -III, a pro-aggregating molecule, is believed to derive from non enzymatic oxidation of arachidonic acid. We assessed the hypothesis that PGF2 α -III derives also from gp91phox activation and contributes to the phenomenon of platelet recruitment. We studied PGF2 α -III in platelets from 8 male patients with hereditary deficiency of gp91phox (Nox2) the catalytic subunit of NADPH oxidase, and 8 male controls. Gp91phox deficency is a very rare human disease(prevalence 1:1000.000) characterized by life-threatening infections. Patients were a subgroup from a multicentre study in collaboration with the Italian Primary Immunodeficiency Network (IPINET). Upon stimulation platelets from healthy subjects produced PGF2 α -III, that was inhibited 8% by aspirin and 43% by an inhibitor of gp91phox. Also, in platelets incubated with a gp91phox inhibitor or with SQ29548, a thromboxane A2 receptors inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 52% and 84% respectively; a lower effect (−13%) was seen with aspirin. Incubation of normal platelets with PGF2 α -III dose-dependently(1–100 pM) increased platelet recruitment by mobilizing platelet Ca2+ and activating gpIIb/IIIa. Platelets from patients with gp91phox hereditary deficiency had normal thromboxane A2 formation and 75% PGF2 α -III reduction compared to controls. In gp91phox-deficient patients agonist-induced platelet aggregation was within the normal range while platelet recruitment was reduced compared to controls. Incubation of gp91phox-deficient platelets with PGF2 α -III (1–100 pM) partially restored platelet recruitment. In conclusion this study provides the first evidence that platelet PGF2 α -III maximally derives from Nox2 activation and contributes to platelet recruitment via activation of gpIIb/IIIa.

Nox2-Containing NADPH Oxidase Deficiency Confers Protection From Hindlimb Ischemia in Conditions of Increased Oxidative Stress

Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2(-/-)). Nox2(-/-) mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2(+/+) mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2(-/-) endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2(+/+) endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.

The NADPH oxidase NOX2 plays a role in periodontal pathologies

Oxidative stress plays an important role in periodontal health and disease. The phagocyte nicotinamide adenine dinucleotide phosphate oxidase NOX2 is most likely one of the key sources of reactive oxygen species (ROS) in periodontal tissues. This review will discuss three clinical aspects of NOX2 function. We will first focus on oral pathology in NOX2 deficiency such as chronic granulomatous disease (CGD). CGD patients are thought to suffer from infections and sterile hyperinflammation in the oral cavity. Indeed, the periodontium appears to be the most common site of infection in CGD patients; however, as periodontitis is also common in the general population, it is not clear to which extent these infections can be attributed to the disease. Secondly, the role of oxidative stress in periodontal disease of diabetic patients will be reviewed. Diabetes is indeed a major risk factor to develop periodontal disease, and increased activity of leukocytes is commonly observed. Enhanced NOX2 activity is likely to be involved in the pathomechanism, but data remains somewhat preliminary. The strongest case for involvement of NOX2 in periodontal diseases is aggressive periodontitis. Increased ROS generation by leukocytes from patients with aggressive periodontitis has clearly been documented. This increased ROS generation is to be caused by two factors: (1) genetically enhanced ROS generation and (2) oral pathogens that enhance NOX function. NOX enzymes in the oral cavity have so far received little attention but are likely to be important players in this setting. New therapies could be derived from these new concepts.

Nox2 Is Determinant for Ischemia-Induced Oxidative Stress and Arterial Vasodilatation: A Pilot Study in Patients With Hereditary Nox2 Deficiency

To the Editor: Reactive oxidant species (ROS) are a family of molecules that are involved in the modulation of arterial tone via rapid degradation of nitric oxide (NO).1 NADPH oxidase is a predominant cellular source of O2−-producing enzymes.1 Four homologs of gp91phox (Nox 2) named Nox1, Nox3, Nox4, and Nox5 have been identified as components of nonphagocyte-type NADPH oxidase.2 Recent studies performed in Nox1 and Nox2 knock-out animals suggested that these Nox isoforms may be implicated in controlling vascular function via modulation of NO bioactivity.3,4 X-chronic granulomatous disease (X-CGD) is a rare primary immunodeficiency affecting the innate immunologic system; it is caused by mutations in any of the 4 genes encoding subunits of the O2− generating NADPH oxidase, resulting in defective O2− generation and intracellular killing.5 We speculated that in patients with downregulation of NADPH oxidase the …

Decreased neointimal formation in Nox2-deficient mice reveals a direct role for NADPH oxidase in the response to arterial injury.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced, in part, from NADPH oxidase in response to host invasion and tissue injury. Defects in NADPH oxidase impair host defense; however, the role of ROS and RNS in the response to tissue injury is not known. We addressed this issue by subjecting leukocyte oxidase (Nox2)-deficient (Nox2-/-) mice to arterial injury. Femoral artery injury was associated with increased Nox2 expression, ROS/RNS production, and oxidative protein and lipid modification in wild-type mice. In Nox2-/- mice, RNS-mediated protein oxidation, as monitored by protein nitrotyrosine content, was significantly diminished. This was accompanied by reduced neointimal proliferation, as monitored by intimal thickness and intimal/medial ratio, in Nox2-/- compared to wild-type mice. In addition, Nox2 deficiency led to reduced cellular proliferation and leukocyte accumulation. These data indicate that Nox2-mediated oxidant production has a requisite role in the response to tissue injury.